Therapeutic Potency of Mono- and Diprenylated Acetophenones: A Case Study of In-Vivo Antimalarial Evaluation

Abstract Malaria remains a febrile infection of public health concern in many countries especially tropical countries in Africa and certain countries in Southern and North America such as Brazil, Costa Rica, Mexico, Dominican Republic, Colombia, and Ecuador. Hence this has made research into this area paramount. Acetophenones are active fragments in many compounds with promising antimalarial activity, such as chalcones. The aim of the present study was to investigate antimalarial activity of 3,5-diprenyl acetophenone ( I ) and 5-diprenyl acetophenone ( II ) in in vivo . In this study, compounds I and II were synthesized using an aromatic substitution reaction. The in-vivo antimalarial potential of compounds I and II was analyzed in Plasmodium berghei -infected mice. Our data showed that compound I (25, 50, and 100 mg/kg) had promising antimalarial activity, with parasitemia inhibited rate being 68.03, 65.16, and 69.75%, respectively. Compound II dose-dependently inhibited parasitemia levels, it demonstrated an infinitesimally higher activity (72.12%) when compared with compound I (69.75%) at 100 mg/kg dose. The two compounds passed the rule of three, Lipinski's rule of five, predicted plausible pharmacokinetic profile (ADME), and apparent safety profile, and demonstrated drug-like fragments. The study provided guidance in exploring novel antimalarial compounds based on the scaffolds of prenylated acetophenones.


Introduction
Malaria is an infectious and life-threatening disease that affects nearly half the population of the world, it is typically found in tropical and subtropical climates where the parasite can grow. Africa is responsible for 90% of worldwide malaria morbidity and death, particularly pediatric mortality. 1 Plas-Several antimalarial drugs have been used to reduce sensitivity of these parasites to these antimalarial drugs. The most potent antimalarial agents are the artemisininbased combination therapies (ACTs); however, with reports of potential resistance to ACTs, there are great concerns among researchers in the field of drug design for potent antimalarial agents against the drug-resistant strains of Plasmodium. Although ACTs remain the first line of defense against malaria, backup or alternative solutions are needed to deal with ACT-resistant strains produced by these parasites. 2 One of the more recent areas of exploration in drug design is the fragment-based drug design, where most active scaffolds or moieties of active drug compounds can be used in designing drug compounds with synergistic-like effects in terms of activity and physicochemical properties such as reduced or less toxic effects caused by a process called molecular hybridization. 1,4 To combat this disease, new antimalarial agents with novel biological targets are required.
Acetophenone is an organic compound that is used in the synthesis of many organic pharmaceuticals, and it is also a precursor for many resins and fragrances. General structure of acetophenone is shown in ►Fig. 1. Proteases form one of the most explored or studied antimalarial mediating targets. [5][6][7][8] And acetophenone is one of the important fragments with protease-inhibiting activity. A condensation of acetophenone and aldehyde gives a very important class of organic compounds known as chalcones. Chalcones offer a wide range of medicinal properties, including antiplasmodial activity. 2 Acetophenones form part of the most active fragment of the chalcones and other classes of organic compounds. Modifications on the acetophenone ring improve its activity. Some acetophenones used in the synthesis of chalcones have been modified through the addition of electron-withdrawing or -donating substituents such as methoxy, hydroxy, chlorine, and alkyl to give different pharmacological activities. The presence of electron-donating groups on the acetophenone ring, according to Li et al, 9 is advantageous to antimalarial activity. Structure-activity relationship (SAR) evaluation shows that the size and hydrophobicity of the substituents attached on the acetophenone are critical parameters for regulating the activity of the ring. 10 In this work, two prenylated acetophenones, 3,5-diprenyl acetophenone (I) and 5-diprenyl acetophenone (II), were chosen to assess their antimalarial activity to correlate the theoretical studies, e.g., rule of three (Ro3) for active fragment and rule of five (Ro5) for orally bioavailable agents, and in silico pharmacokinetic (PK) studies with their experimental activity, and deduce further insights into their SAR. This is the first report of the in vivo antimalarial activity of the two prenylated acetophenones.
The melting points of the compounds were measured without correction using the Gallenkamp melting point instrument. Experiments of proton nuclear magnetic resonance ( 1 H-NMR) and carbon-13 nuclear magnetic resonance ( 13 C-NMR) were conducted in the Department of Chemistry, University of Kwazulu-Natal, South Africa, and Multi-user Laboratory, Ahmadu Bello University, Zaria, using 400 MHz Bruker and 400 MHz Agilent, respectively. Chemical shift (d) in ppm downfield from tetra-methyl silane as the internal standard was used to capture the nuclear magnetic resonance data. Wavenumbers were captured as Fouriertransform infrared spectroscopy (FTIR) using an Agilent spectrophotometer (cm À1 ).

Synthesis of Prenylated 2,4-Dihydroxyacetophenone
The synthesis of compound I, II, and III was described in ►Fig. 2. The two prenylated acetophenones (I and II) were synthesized through an aromatic electrophilic substitution reaction. Equimolar quantities of 3-methyl-2-buten-1-ol and boron trifluoride diethyl etherate catalyst (20 mmol) were dissolved in anhydrous 1,4-dioxane to acquire a pink-red solution, to which 20 mmol of 2,4-dihydroxyacetophenone was subsequently added. The solution was continuously stirred for 2 hours. This reaction was performed at room temperature under inert conditions (nitrogen atmosphere). Thin-layer chromatography profile was used to monitor the progress of the reaction. On completion of the reaction, the mixture was diluted with diethyl ether (100 mL) and washed with water (50 mL Â 3). The organic phase was dried over anhydrous Na 2 SO 4 and evaporated under reduced pressure to obtain a residue, which was purified by a column chromatography using n-hexane and n-hexane/ethylacetate (7:3) to afford compounds I, II, and trace amount of compound III.

Animals
Forty locally bred adult Swiss Albino mice, weighing 18 to 22 g, were housed in conventional laboratory settings with unlimited access to pellet feed and water ad libitum. The animals were housed in clean polypropylene cages at the Department of Pharmacology and Therapeutics' Animal House. All animal experiments followed Ahmadu Bello University's research policy and guidelines for the use and care of laboratory animals, which are widely acknowledged globally. Ethical approval was sought and obtained from ABU Committee on Animal Use and Care.

Malarial Parasite
Malaria parasites (Plasmodium berghei NK 65) were provided by the Department of Pharmacology and Therapeutics, Ahmadu Bello University, Zaria. The mice were inoculated intraperitoneally with 0.2 mL standard inoculum containing approximately 10 7 parasitized red blood cells.

Evaluation of Theoretical Oral Bioavailability, Pharmacokinetics, and Toxicity
Prior to synthesis, the oral bioavailability of the two prenylated acetophenones was theoretically predicted using Lipinski's Ro5 on SWISS ADME web tools. (http://www. swissadme.ch). Ro3 was used to predict the propensity of   their activity. In silico PK study and toxicity evaluation was performed on Admetlab2.0 and Protox-II.

Inoculation of Plasmodium berghei Parasite
Infected blood was taken from the tail vein of a donor mouse with a parasitemia level of 20 to 25% using heparinized capillary tubes and then transferred to a sterile plain beaker. Two milliliters of blood were diluted with 10 mL of normal saline, yielding 0.2 mL of infected red blood cells. The mice were then given 0.2 mL of blood suspension intraperitoneally.

Treatment
To test the curative efficacy of the synthesized compounds against an established plasmodium infection, Ryley and Peters' approach was used. 11 The mice were infected with the parasite as described by Ryley and Peters' method and were left untreated for 72 hours, following which parasitemia levels were estimated to be between 20 and 25%. 11 The mice with the required parasitemia levels were split into eight groups (n ¼ 5 in each group). Group 1 was given the negative control intraperitoneally [1% (w/v), acacia (Sigma Aldrich)]. Groups 2 to 7 were given 25, 50, and 100 mg/kg of test compounds, while group 8 was given 5 mg/kg of the positive control [chloroquine (Jiangsu Ruinian Quianjin Pharmaceutical Co. Ltd.)]. All treatments were given intraperitoneally for 4 days consecutively. On the fifth day, blood was obtained from the tail vein of the treated mice from all groups. A thin film was made by smearing the blood samples on microscopic slides. The slides were fixed in absolute methanol and stained with 3% Giemsa solution at pH 7.2. Average levels of parasitemia were calculated from six different fields. The average suppression percentage of the parasite was calculated for each group using Equation (1): where A is average levels of parasitemia of negative control; B is average levels of parasitemia in each treated group.

Statistical Analysis
The data were analyzed with SPSS 20.0 software and displayed as mean AE standard error of the mean. The mean difference between the results obtained was compared using a one-way ANOVA (analysis of variance) followed by Dunnett's post-hoc test. Statistical significance was defined as a p-value of less than 0.05.

Drug-Likeness
Compound II efficiently passed Ro3 for active fragments having molecular weight 300 g/mol, MlogP 3, number of hydrogen bond acceptor 3, number of hydrogen bond donor 3, number of rotatable bonds 3, polar surface area 60 Å, while compound I passed Ro3 on the average according to results in ►Table 1. Ro3 has been useful in ensuring that fragment libraries really do consist of compounds with active fragment-like properties. Any compound that passes the Ro3 on average could be useful when constructing fragment libraries for efficient lead discovery. As shown in ►Table 2, both compounds passed the Lipinski's Ro5 as indicated in the reported studies, 12,13 suggesting that they have high probability of being orally bioavailable.

Absorption
As shown in ►Table 3, compounds I and II had optimal and medium permeability in both in-vivo and in-vitro predictive models, respectively. The in-vivo permeability of compound II in the predictive human colon adenocarcinoma cell lines (Caco-2) was slightly higher when compared with compound I, with values of being À4.64 and À4.70 cm/s, respectively. However, permeability co-efficient (Papp) values of both compounds were 15 Â 10 À6 cm/s, which is greater than 2 Â 10 À6 cm/s but less than 20 Â 10 À6 cm/s in Madin À Darby canine kidney cells (MDCKs), suggesting that they had medium permeability in MDCK model.
Compound I had strong substrate affinity, while poor inhibitory affinity for P-gp enzyme with scores of 0.05 and 0.82, respectively. On the other hand, compound II had comparable substrate affinity for P-gp with scores of 0.02 and stronger inhibitory affinity for P-gp with scores of 0.07, respectively. The observed disparities between Caco-2 permeability and MDCK permeability for both compounds can be explained by P-gp inhibitory and substrate affinities. Drugs that are P-gp substrates usually have disparities in their Caco-2 and MDCK permeability. An instance is quinidine, a P-gp substrate that had high permeability in the Caco-2 model but medium permeability in the MDCK model. This explained why both compounds being P-gp substrates with comparable affinities indicated high permeability in the Caco-2 model and medium permeability in the MDCK model. 14 Compounds I and II had high human intestinal absorption with scores of 0.02 and 0.01, suggesting that more than 30% of the compounds is absorbed in human intestine. Also, the result showed that compound II is more readily absorbed in the human intestine in comparison to compound I.
Oral bioavailability prediction suggested that the two compounds are orally bioavailable complementing the results of Lipinski's rule's results. Compounds I and II had

Distribution
As shown in ►Table 4, compound I was predicted to have protein plasma binding (PPB) of 89.96%, a value considered as optimal PPB, and compound II predicted a higher PPB value of 97.49%. However, statistics have shown that lump-sums of United States Food and Drug Administration (FDA)-approved drugs have PPB greater than 99%. 14 The volume distribution (VD) values of both compounds fall between 0.04 and 20 L/kg for optimal distribution as indicated by the results in ►Table 4. As a consequence of their PPB values, compound I demonstrated medium fraction unbound (F u ) of 7.638% while compound II indicated a low F u of 3.974%. In spite of their low F u , both compounds had excellent blood-brain barrier penetration with scores less than 0.1.

Metabolism
Compound I is a strong inhibitor and substrate of CYP2D6 and CYP3A4, a weak inhibitor of CYP2C9, and a noninhibitor of CYP1A2 and CYP2C19. Also, the compound has high substrate affinity for CYP1A2 and CYP2C19, and was nonsubstrate of CYP2C9 enzyme (►Table 5). On the other hand, compound II was a strong inhibitor and substrate of CYP3A4, a weak inhibitor of CYP2C9, and a noninhibitor of CYP2D6, CYP1A2, and CYPC19 enzymes. Compound II is also a strong substrate of CYP2C19, a weak substrate of CYP1A2 and CYP2D6, and a nonsubstrate of CYP2C19 (►Table 5).

Organ Toxicity
Lethality dose (LD 50 ) of compounds I and II were 2,830 mg/kg (►Table 7), which were categorized as Class V: (2,000 mg/kg < LD 50 5,000 kg/kg), suggesting that they may be harmful if swallowed according to the toxic class of the globally harmonized system of classification of labeling of chemicals. Predicted rat or mice oral acute toxicity (OAT) scores of compounds I and II were 0.21 and 0.07, which fall in the category of OAT >500 mg/kg for low toxicity translating that the compounds are safe. Our data also showed that compounds I and II are predicted as noncauser of human hepatotoxicity or drug-induced liver injury with inactivity probabilities of 0.68 and 0.57, noncarcinogenic with probabilities of 0.68 and 0.70, nonimmunotoxic with probabilities of 0.99 and 0.62, nonmutagenic with probabilities of 0.75 and 0.75, and noncytotoxic with probabilities of 0.76 and 0.71, respectively (►Table 7).
Furthermore, compounds I and II are also nonblocker of the human ether-a-go-go related gene (hERG) as indicated by their scores of 0.01 (►Table 8), which translates to an excellent safety profile. Therefore, they may not cause hERG toxicities which include long QT syndrome, arrhythmia, and Torsades de Pointes that were associated with palpitations, fainting, or even sudden death. 14 Compound I could not cause respiratory toxicity with excellent safety score of 0.16, while compound II has a score of 0.40 (►Table 8), suggesting a lower safety profile when compared   with compound I. Also, both compounds are nontoxic, with FDA maximum recommended daily dose below 0.2 mmol/kg-bw/d (►Table 8). However, compound I was predicted to be skin sensitive with a score of 0.84, therefore may not be formulated for topical application. While compound II was relatively non-skin sensitive with a score of 0.53, it may be formulated for topical application. As shown in ►Table 9, compounds I and II are non-eye corrosive with excellent safety scores of 0.004 and 0.06, respectively. However, compound II is an eye irritant with        a score of 0.94, while compound I is relatively non-eye irritant with a score of 0.47.

Tox21 Pathway
Nuclear receptor pathway toxicity. Compounds I and II was predicted not to interact with any of the nuclear receptors with probabilities between 0.85 for estrogen receptor (NR-ER) and 0.99 for androgen receptor (NR-AR) according to results in ►Table 10. This suggests that the compounds may not cause nuclear receptor pathway toxicity. Stress response pathway toxicity. Compound I was predicted as nontoxic to stress response pathways indicating noninteraction with any of the stress response receptors. The noninteraction probabilities of the compound ranged between 0.76 for mitochondrial membrane potential (SR-MMP) to 0.97 for ATPase family AAA domain-containing proteins 5 (SR-ATAD5) (►Table 11). While compound II was also inactive against nearly all of the stress response receptors with noninteraction probabilities between 0.77 for phosphoprotein (tumor suppressor) p53 SR-p53 and 0.97 for SR-ATAD5 (►Table 11). It is found to interact with SR-MMP with 0.61 activity probability.
Environmental toxicity. Compounds I and II had bioconcentration factors (BCFs) <3.000 log10(L/kg) according to the results in ►Table 12, which corresponds to BCF <1,000 L/kg categorized as nonbioaccumulative by the United States Environmental Protection Agency under the Toxic Substances Control Ac. These values are also below the 3.700 earmarked by Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) threshold for very bioaccumulative chemicals. While compounds I and II had equal tetrahymena pyriformis (IGC 50 ) values (3.77). Compound I demonstrated safer fathead minnow LC 50 (LC 50 FM, 5.12) and daphnia magna LC 50 (LC 50 DM, 6.13) when compared with compound II (4.44 and 5.31, respectively).

Antimalarial Activity
Mice with plasmodium infection were treated with compound I (25, 50, 100 mg/kg) and compound II (25, 50, 100 mg/kg). Chloroquine was used as a control drug. Then, parasitemia levels were evaluated, and the results are shown in ►Table 13. Our data showed that parasitemia levels in mice treated with compounds I and II were significantly lowered than those treated with distilled water, suggesting the antimalarial activity of the two compounds; however, parasitemia levels of compound I and II-treated group were much higher than the control drug (chloroquine), suggesting a much weaker activity of the two compounds when compared with the reference drug.

Synthesis and Characterization
Two prenylated acetophenones were synthesized using 3methyl-2-buten-1-ol under nitrogen conditions to minimize oxidative side-product(s) and efficient prenylation on positions 3 and 5 of the dihydroxy acetophenone. The synthesis was achieved in low yields of 6.25, 10.0, and 0.07% for compounds I, II, and III, respectively. Oral bioavailability is an important parameter in drug design as it reduces drug failure resulting from poor PK profile. The two compounds passed Lipinski's rule as revealed in ►Table 2. This showed that the compounds would be orally bioavailable.
Compounds I, II, and III were found to be white crystalline solids according to a reported study. 15 Melting points were within the range of 110 to 112°C for compound I, 145 to 146°C for compound II, and 154 to 156°C for compound III. The FTIR spectra showed the presence of prominent bands at 3,384-3,272 cm À1 (OH stretching vibration), 1,613-1,617 cm À1 (C ¼ O stretching vibration), 2,851-2,728 cm À1 (sp 3 C-H stretching vibration), 2,914-2,911 cm À1 and 2,974-2,967 cm À1 (sp 2 C-H stretching vibration) (►Figs. [3][4][5]. The sp 2 and sp 3 stretches indicated the presence of the prenylated groups on compounds I, II, and III. The 1 H NMR and 13 C NMR spectra were used to elucidate the structures of compounds I and II. Also as shown by 1 H and 13 C NMR spectra, there were appearances of distinct peaks indicating prenylation had occurred on the substituted acetophenone ring. The 1 H-NMR of compound I showed distinct peaks at 5.31 and 6.27 ppm, indicating the presence of phenolic protons on the aromatic group, peaks at 3.33 and 3.23 ppm corresponded to allylic protons in the prenylated groups. Peaks at 7.49 ppm indicated the aromatic proton. Peaks at 1.76 ppm corresponded to signals for terminal methyl groups of the prenyl groups, while the peak at 2.50 ppm corresponded to α-methyl of the ketone. For compound II, the proton peaks at 7.58 and 5.51 ppm corresponded to aromatic protons, the peaks at 5.37 and 5.11 ppm indicated for phenolic protons, the peak at 3.54 ppm indicated for allylic proton, the peak at 3.45 ppm corresponded to methylene protons of the prenyl group, the peak at 2.69 ppm corresponded to α-methyl of the ketone, and the peak at 1.751 ppm indicated for the terminal methyl groups on compound II accordingly.

Antimalarial Activity
This study is the first report of antimalarial activity of prenylated acetophenone. As shown in ►Table 13, at doses of 25 and 50 mg/kg, compound I showed promising activity with percentage inhibition of 68.03 and 65.16% respectively, demonstrating superior activities when compared with compound II (33.33 and 39.43%). This suggests that prenylation at position 3 of the acetophenone is important for antimalarial activity. Furthermore, the superior activities of compound I may be due to its better PK profiles in respect to their PPB, VD, and F u as displayed in ►Table 4. However, at a higher dose of 100 mg/kg, compound II demonstrates supper-activity with the inhibition rate of parasitemia level being 72.16% when compared with compound I (69.75%). Furthermore, the antimalarial activity of compound II was dose-dependent. A closer analysis also indicated 100% increase in activity of compound II when the dose was increased from 50 to 100 mg/kg.
To evaluate the possible prodrug effect of the compounds, the curative model was chosen for the study. Krettli et al suggested that a compound should be considered active when its parasitemia reduction is !30%. 16 In another study, antimalarial agents are classified into three categories as moderate, good, and very good if the compound showed parasitemia suppression percentage equal or greater than 50%. 17,18 Consequently, based on these criteria, compounds I and II were presumed to have very good antimalarial activity. Furthermore, compounds I and II demonstrated a superior antimalarial activity compared with nerolidylcatechol and its derivatives in a similar study, 19 suggesting that the resorcinol moiety of compounds I and II is important for antimalarial activity and therefore confers more antimalarial activity compared with the catechol moiety. Early communication of these findings has been reported in a preprint. 20

Conclusion
The two prenylated compounds showed significant antimalarial activity as displayed by their ability to suppress Plasmodium berghei infection in mice. The two prenylated acetophenones demonstrated drug-like physicochemical parameters with good PK profile and excellent safety profile. Thus, these scaffolds should play significant roles in designing compounds with better antimalarial activity.

Supporting Information
Proton and carbon NMR of compound I and II are included in the ►Supporting Information (►Figs. S1-S4 [online only]).

Conflict of Interest
The authors declare no conflict of interest.